This invention relates to an antigenic preparation and in particular to the use of this antigenic preparation for the treatment and prevention of gastroduodenal disease associated Helicobacter infection, particularly with Helicobacter pylori infection in humans.
Helicobacter pylori is a bacterium that infects the stomach lining of perhaps half the world""s population. Infection with the organism is usually chronic, and results in continuing inflammation of the gastric mucosa. The infection is often asymptomatic. However, in association with other cofactors, a proportion of infected people go on to develop sequelae including peptic ulceration of the stomach or duodenum, gastric adenocarcinomas and lymphomas. Peptic ulcer treatment studies have shown that cure of H. pylori infection is associated with a dramatic reduction in the relapse rate of this usually chronic disease. Long term infection with H. pylori leads to the development of chronic atrophic gastritis, which has long been recognised as a precursor lesion in the development of gastric cancer. Thus a number of studies have now linked preceding H. pylori infection with an increased risk of developing gastric cancer. Therefore the treatment and prevention of H. pylori infection has the potential to prevent considerable mortality and morbidity from gastroduodenal disease.
There is no laboratory animal model of H. pylori infection suitable for use in screening new therapies and vaccines for H. pylori infection. However, a Helicobacter felis mouse model of gastric Helicobacter infection has been developed that has proved extremely useful in the screening new antimicrobial therapeutic regimens and vaccination protocols (Lee et al. 1990; Dick-Hegedus and Lee, 1991). H. felis is a spiral shaped bacterium that is very closely related to H. pylori. This bacterium colonises the stomach of mice in a very similar way to H. pylori in the human i.e. the main ecological niche is gastric mucus and colonisation is mainly seen in the antrum of the stomach. In germfree mice, H. felis infection induces a gastritis that is very similar to the human H. pylori infection with a chronic inflammation of mononuclear cells accompanied by a polymorphonuclear leucocyte infiltration. Infection with either organism results in the induction of a similar raised systemic humoral immune response against H. pylori and H. felis respectively (Lee et al., 1990).
The H. felis mouse model has proved to be very predictive of the efficacy of anti-H. pylori agents in humans. Thus, monotherapy with agents with high in vitro activity such as erythromycin show no significant in vivo effect against H. felis in mice, just as erythromycin has no anti-H.pylori effect in humans, despite its high antimicrobial effects in vitro. In contrast, the triple therapy regimens of a bismuth compound, metronidazole, and tetracycline or amoxycillin lead to a very high eradication rate in H. felis infected mice (Dick-Hegedus and Lee, 1991). Such triple therapies are the most successful human anti-H. pylori regimens, and at the present time are recommended as the first choice for anti-H. pylori therapy. The H. felis mouse model has also been used to demonstrate that mice can be orally immunised with Helicobacter antigen preparations of sonicated cells and cholera toxin adjuvant, to both treat active H. felis infection and to protect against H. felis infection, however the protective antigen or antigens in these preparations was not determined (see International Patent Application No. PCT/AU94/00416; Czinn et al., 1993).
In work leading to the present invention, a protective antigen of Helicobacter organisms has been identified, this antigen being recognised by monoclonal antibodies which are effective in treatment of H. felis-infected mice.
In one aspect, the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection, which comprises the lipopolysaccharide (LPS) of Helicobacter bacteria, or an immunogenic fragment thereof.
Preferably, the antigenic preparation comprises an at least partially purified LPS preparation.
The term xe2x80x9cat least partially purifiedxe2x80x9d as used herein denotes a preparation in which the LPS content is greater, preferably at least 30% and more preferably at least 50% greater, than the LPS content of a whole cell sonicate of Helicobacter bacteria. Preferably, the preparation is one in which the LPS is xe2x80x9csubstantially purexe2x80x9d, that is one in which the LPS content is at least 80%, more preferably at least 90%, of the total Helicobacter antigens in the preparation.
It is to be understood that the present invention extends not only to an antigenic preparation comprising the LPS of Helicobacter bacteria, but also to antigenic preparations comprising immunogenic fragments of this lipopolysaccharide, that is LPS fragments which are capable of eliciting a specific protective immune response in a mammalian host. Such immunogenic fragments may also be recognised by Helicobacter-specific monoclonal antibodies, particularly monoclonal antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.
In another aspect, the present invention provides a vaccine composition for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an immunologically effective amount of an antigenic preparation as broadly described above, optionally in association with an adjuvant, together with one or more pharmaceutically acceptable carriers and/or diluents.
In yet another aspect, the present invention provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of an immunologically effective amount of an antigenic preparation as broadly described above, optionally in association with an adjuvant.
In a related aspect, this invention provides the use of an immunologically effective amount of an antigenic preparation as broadly described above, optionally in association with an adjuvant, for the treatment or prevention of Helicobacter infection in a mammalian host.
In yet another aspect, the invention provides the use of an antigenic preparation as broadly described above, optionally in association with an adjuvant, in the manufacture of a vaccine composition for the treatment or prevention of Helicobacter infection in a mammalian host.
Preferably, but not essentially, the antigenic preparation of this invention is orally administered to the host, and is administered in association with a mucosal adjuvant. However, the invention also extends to parenteral administration of this antigenic preparation.
The present invention also extends to an antibody, particularly a monoclonal antibody, specific for the antigenic preparation as broadly described above, and in particular specific for the LPS of Helicobacter bacteria, including H. felis LPS and H. pylori LPS.
In this aspect, the invention further provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises passive immunisation of said host by administration of an effective amount of an antibody, particularly a monoclonal antibody, specific for the antigenic preparation as broadly described above.
By use of the term xe2x80x9cimmunologically effective amountxe2x80x9d herein in the context of treatment of Helicobacter infection, it is meant that the administration of that amount to an individual infected host, either in a single dose or as part of a series, is effective for treatment of Helicobacter infection. By the use of the term xe2x80x9cimmunologically effective amountxe2x80x9d herein in the context of prevention of Helicobacter infection, it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, is effective to delay, inhibit or prevent Helicobacter infection. The effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual""s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, is to be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Preferably, the antigenic preparation of this invention comprises a preparation of the LPS of H. pylori or H. felis. Preferably also, this antigenic preparation is used in a vaccine composition for oral administration which includes a mucosal adjuvant.
In a particularly preferred aspect of this invention, an oral vaccine composition comprising an antigenic preparation of at least partially purified H. pylori LPS in association with a mucosal adjuvant is used for the treatment or prevention of H. pylori infection in a human host.
The mucosal adjuvant which is optionally, and preferably, administered with the at least partially purified Helicobacter LPS preparation to the infected host is preferably cholera toxin. Mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the Helicobacter LPS preparation. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin. Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.
The Helicobacter LPS preparation may be delivered in accordance with this invention in ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycodside) to form microspheres of a size suited to adsorption by M cells. Alternatively, micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors. The Helicobacter LPS preparation may also be incorporated into oily emulsions and delivered orally. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter, (1992).
Other adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in the prophylactic or therapeutic vaccine composition of this invention. The vaccine composition may, for example, be formulated in enteric coated gelatine capsules including sodium bicarbonate buffers together with the Helicobacter LPS preparation and cholera toxin mucosal adjuvant.
The formulation of prophylactic or therapeutic vaccine compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions of the present invention is contemplated.
The Helicobacter LPS preparation of the present invention may be administered as the sole active immunogen in a vaccine composition. Alternatively, however, the vaccine composition may include other active immunogens, including other Helicobacter antigens, as well as immunologically active antigens against other pathogenic species.
It is especially advantageous to formulate the vaccine compositions of this invention in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
In work leading to the present invention, the H. felis model described above was used to examine the immune response in immunised and protected mice by generating hybridoma cell lines from fusions of SP-2 cells and lymphocytes from immune mice. These hybridoma cells were screened for the production of monoclonal antibodies that recognised H. felis. A hybridoma cell line secreting IgA antibodies that recognised and agglutinated H. felis was selected and used to treat infected mice. A significant proportion of these mice were cured of their infection. Thus the antigen recognised by the monoclonal antibody was identified as a protective antigen.
This protective antigen was then identified by biochemical methods as the lipopolysaccharide (LPS) constituent of the outer membrane of the bacteria. The close relationship between H. felis and H. pylori clearly indicates that, as H. felis LPS has been identified as a protective antigen in the mouse model and as vaccination with H. pylori organisms protects mice against subsequent H. felis infection, then H. pylori LPS will be a protective antigen in the human infection. Accordingly, antibodies raised in a mammalian host against the Helicobacter LPS preparation by active vaccination will have the ability to eradicate or at least suppress an existing infection, or prevent establishment of a new infection, in this host. Furthermore, passive immunisation of a mammalian host with antibodies raised against the Helicobacter LPS preparation will similarly eradicate or at least suppress an existing infection, or prevent a new infection, in the host.
Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.